Technical Abstract:
Tomato spotted wilt virus (TSWV) has one negative sense (L) and two ambisense (M and S) RNAs. The lack of a tractable TSWV reverse genetics system has impeded direct demonstration of gene function and prevented the usual types of pathogen characterization. The TSWV NSm (non-structural) protein has been presumed for several years to be the viral movement protein (MP) based on a few lines of indirect evidence. We determined that a Florida field isolate of TSWV could complement local, and to a lesser extent, systemic movement of a movement-defective Tobacco mosaic virus (TMV) expression vector in Nicotiana benthamiana. To determine if NSm was responsible for this complementation, we cloned the NSm open reading frame (ORF) from this isolate and constructed TMV hybrids expressing the NSm ORF to test whether this tubule forming, putative MP could functionally substitute for the TMV MP protein. Initially, the NSm ORF was expressed from either the MP or coat protein subgenomic (sg) promoter of TMV. Both of these TSWV-TMV hybrids replicated in tobacco suspension cells and directed expression of NSm. Both of these free-RNA hybrids were restricted to inoculated leaves of Nicotiana tabacum cv. Xanthi but moved into upper leaves of N. benthamiana, directly demonstrating that NSm is the TSWV MP. To monitor movement in plants, the ORF for the jellyfish green fluorescent protein (GFP) was inserted behind an additional sg promoter downstream of NSm. These TSWV-TMV hybrids moved and expressed both NSm and GFP in plants. These results demonstrate the utility of a well characterized viral genetic system to dissect gene functions for a virus lacking a reverse genetics system and provide a means to explore the function of TSWV genes in tomato and other economically important crops.